Several publications are referenced throughout the specification to describe the state of the art to which this invention pertains. These publications are incorporated by reference herein.
Plant disease is a major cause of crop loss. Various strategies have been developed to control disease, one of the most common of which is the use of chemicals. This approach is usually expensive, not always effective, and often harmful to humans and the environment. A preferred approach is to develop, through breeding, genotypes resistant to diseases.
Conventional plant breeding has been based on genetic recombination through sexual crosses. Although nearly all of the current improved plants are developed through conventional breeding techniques, there are limitations to this approach. These include the lack of availability of appropriate germplasm and the incompatibility of certain crosses.
With the recent advances in molecular biology and gene transfer technology for plants, it has become possible to circumvent some of the limitations of conventional breeding by obtaining genes of interest from diverse sources and introducing them into crop plants by genetic transformation. Methods are now available in the art for transforming a wide variety of plant species, including both monocotyledonous and dicotyledonous flowering plants, which contain nearly all agronomically and horticulturally important crops.
The microbial causal agents of plant disease include fungi, bacteria and viruses. Plants have active mechanisms for defending themselves against microbial pathogen infection. For most resistant plants, challenge by a pathogen results in induction of a dual defense mechanisms, part of which is located at the site of infection, occurring as necrotic lesions resulting from host cell death (hypersensitive responses). Another part of the defense occurs in the surrounding, and even distal, uninfected parts of the plant (systemic acquired resistance). The HR is a highly regulated process involving cellular protein synthesis, increased cytosolic calcium ion levels, generation of reactive oxygen species, alteration of protein phosphorylation, among other signal responses. Both the hypersensitive response and systemic acquired resistance are associated with activated expression of a large number of defense or defense-related genes (such as PR genes), some of whose products may play important roles in the restriction of pathogen proliferation and spread by participating in strengthening host cellular structures or through their direct antimicrobial activities.
Salicylic acid has been identified as an important signalling factor in the induction of plant disease resistance. Evidence indicates that a systemic increase in salicylic acid is important for the induction of systemic acquired resistance. Salicylic acid also appears to play an important role in the primary, local defense associated with the hypersensitive cell death. One proposed mechanism of action of salicylic acid is to inhibit catalase activity, thereby elevating H.sub.2 O.sub.2 levels. These elevated levels of H.sub.2 O.sub.2 or other reactive oxygen species derived from H.sub.2 O.sub.2 may serve as a signal for activation of plant defenses such as the synthesis of PR proteins (Chen et al., 1993, Science 262: 1883-1886).
In some resistance responses, plants challenged with a pathogen produce their own anti-microbial substances, termed "phytoalexins." The production of phytoalexins in the resistance response has been well characterized in several species. For instance, localized and systemic resistance of certain potato varieties to Phytophthora infestans may be elicited by several substances, including various portions of the fungus itself, and certain long-chain polyunsaturated fatty acids, such as arachidonic and eicosopentanoic acids. Systemic resistance of potato plants to P. infestans following surface applications of long-chain polyunsaturated fatty acids, eicosopentanoic (20:5), arachidonic (20:4), linolenic (18:3) and linoleic (18:2), has been reported (Cohen et al., 1991, Physiological and Molecular Plant Pathology 38: 255-263). However, systemic resistance was directly correlated with phytotoxicity of the fatty acids tested (oleic acid (18:1) was found to be neither phytotoxic nor an inducer of systemic resistance). Thus, it would appear from this report that long-chain polyunsaturated fatty acids are unsuitable for purposes of eliciting pathogen resistance in plants, due to phytotoxicity.
In plants, fatty acids are first produced in saturated forms. They are subsequently desaturated by a series of desaturases, the first of which is a .DELTA.-9 desaturase that produces monounsaturated fatty acids. Further desaturation results in the formation of polyunsaturated fatty acids. A .DELTA.-9 desaturase from yeast has been expressed in tobacco (Polashok et al., 1992, Plant Physiol. 100: 894-901) and tomato (Wang et al., 1996, J. Agric. Food Chem. 44: 3399-3402). Expression of yeast desaturase was reported to alter the fatty acid composition of both plants (increasing 16:1 and 18:1 monounsaturated fatty acids, as well as certain polyunsaturated fatty acids), with no apparent alteration in phenotype. However, no reference has been made to the relative pathogen resistance of the yeast desaturase-transgenic plants, as compared to non-transformed plants.